EP2935561B1 - Procédé de récupération de composants intracellulaires à partir de micro-organismes fermentés - Google Patents

Procédé de récupération de composants intracellulaires à partir de micro-organismes fermentés Download PDF

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EP2935561B1
EP2935561B1 EP13826947.7A EP13826947A EP2935561B1 EP 2935561 B1 EP2935561 B1 EP 2935561B1 EP 13826947 A EP13826947 A EP 13826947A EP 2935561 B1 EP2935561 B1 EP 2935561B1
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suspension
nebulization
lysing
solvent
microorganisms
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EP2935561A1 (fr
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Roberta Miglio
Alfredo Montini
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Eni SpA
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Eni SpA
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/48—Separation; Purification; Stabilisation; Use of additives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C12N1/066—Lysis of microorganisms by physical methods
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil

Definitions

  • the present invention relates to a method for recovering intracellular components of materials of a biological origin, such as yeasts, algae, bacteria and/or mildews.
  • suspensions of cellular microorganisms can be lysed by perturbation and breakage of the cell membrane for example by variations in the osmotic pressure.
  • the simple lowering of the ionic force of the liquid environment, for example, or the addition of a surfactant with moderate stirring of the suspension allows the cells to be swollen and allowing them to be lysed.
  • a vigorous stirring of the suspension is necessary, together with a strong thermal, mechanical or sonic action for disrupting the cell membranes, with an increase in the costs of the cell lysis.
  • the cells of bacteria, yeasts and algae, for example are much more resistant and their lysis for recovering the intracellular components is difficult so that resort must be made to more effective methods than osmosis for obtaining them.
  • An industrial plant can generally comprise more kneaders arranged in series (in this case often superimposed in order to limit the encumbrance space) or in parallel, mechanically charged, by means of a hydraulic system, with the oil paste leaving the crusher.
  • This process produces a bland thermal lysis which, associated with a slow remixing of the paste, allows the oily phase to be released, which is then separated by floatation process.
  • This technology is effective for micro-organisms having dimensions larger than a few tens of microns, but it is not effective for resistant micro-organisms such as some, but not only, of the Lypomyces, Rhodotorula and Cryptococcus species.
  • the technical parameters indicated for this process are the temperature and duration of the operation. The temperature is fundamental for the yield in the subsequent extraction and is strictly associated with the releasing phase of the cell content.
  • the time parameter is correlated with the operative mode.
  • Another type of industrial machine capable of effecting this process for cases of cell lysis which require drastic temperature and pressure conditions is a machine consisting of a fixed or rotating cylinder, equipped with internal blades or mixers capable of mixing, breaking and accompanying the cells to be lysed.
  • This machine is used in the food industry for thermal hydrolysis and, at times in acid environments for sulfuric acids (see for example Anco-Eaglin Inc machines).
  • thermomechanical cell lysis acts as a screw press for pressures greater than 5 bar and temperatures higher than 120°C.
  • this machine can also carry out a separation of the cellular liquids during the thermal lysing process for cellular products with membranes characterized by a strong thermal/mechanical breaking strength.
  • glass or ceramic ball mills allows a vigorous shaking and breakage of the cells in the paste to be lysed.
  • the method has been used for years and is considered as being applicable to the biological group of yeasts and fungi, microbacteria, spores and micro-algae. It has more recently been applied to samples of soil and small samples of vegetables and animal tissue.
  • the intense sonication of liquids for example, generates sound waves which propagate in liquid mediums with alternating high- and low-pressure cycles.
  • a high intensity of small-sized vacuum bubbles is created in the liquid.
  • a cavitation process is therefore used for the implosion of the liquid bubbles, which thus allows shear forces to be generated which are capable of mechanically breaking the cell structure.
  • This effect is used for the extraction of lipids from algae.
  • the addition of a solvent is required, as otherwise the cell debris would also remain englobed in the oily/lipid phase.
  • Other methods comprise the crushing of cells by means of high-pressure passage (20,000-30,000 psi or 140-210 MPa) through a valve having a narrow orifice.
  • the fluid is therefore subjected to high shear forces with a consequent breakage of the cells.
  • the shear force for optimizing the breakage of the cell is defined by controlling the pressure.
  • the system requires high energies and a cooling for samples that require multiple steps through the system.
  • the homogenizing valves are dimensioned for obtaining the desired micronization and dispersion degree at the lowest possible pressure, depending on the various applications. They are continuous industrial machines which can also manage voluminous flow-rates with low specific energies of use (for example, a machine which handles liquid flow-rates of 6 m 3 /h, with overpressures of 1,500 bar, requires a power equal to 315 kW).
  • International patent application WO 99/15638 relates to a method for modifying the structure of lipid membranes such as double lipid layers of micro-organisms, eukaryotes or prokaryotes, liposomes, vesicles or living cells.
  • this method creates an instability (perturbation) of the lipid structure which allows the fusion of the lipid membranes without lysing the cells involved.
  • WO 99/15638 provides for the use of a low-temperature spray dryer) for atomizing into small drops a liquid medium such as, for example, a physiological solution, milk, blood, serum, fermentation broths and water, containing the lipid structure, which are introduced at high velocity into an environment containing vapour at a controlled temperature.
  • a liquid medium such as, for example, a physiological solution, milk, blood, serum, fermentation broths and water, containing the lipid structure, which are introduced at high velocity into an environment containing vapour at a controlled temperature.
  • the liquid medium used for suspending the cells or lipid structures to be fused during the nebulisation favours the fusion process of the cell membranes by lysis on the part of these.
  • the liquid medium used must consequently not favour the breakage of these membranes but their aggregation.
  • the objective of the process of WO 99/15638 is not to destroy the membranes of two or more different cell entities, but to keep the characteristics of the original cells unaltered, thus allowing a partial fusion of the cell membranes. In this way, the method produces molecular aggregates which combine the characteristics of the aggregated cells in the multicellular mixture.
  • a lysing or breakage must firstly be effected, for example thermally, in a process volume, or reactor, suitable for obtaining temperatures higher than 120°C, at a pressure of at least 5 bar, for a time ranging from 10 minutes to 4-5 hours under moderate stirring, i.e. the conditions necessary for the lysis of the micro-organisms.
  • This method is characterized in that over 90% of the energy requirements of the overall extraction process of the product of the fermentation process is due to the thermal lysis effected on the fermented micro-organisms.
  • WO 2010/015398 discloses the cell lysis of plant or animal starting material by a combination of a spray method and decompression for the selective extraction and separation of valuable intracellular materials.
  • the present invention relates to a thermal lysing method for the recovery (extraction) of intracellular components of materials of a biological origin, such as cultivated yeasts, algae, bacteria and/or mildews, or in particular, fermented products, which allows the operating costs and costs of the equipment used in the lysing process of said bio-industrial processes, to be minimized.
  • materials of a biological origin such as cultivated yeasts, algae, bacteria and/or mildews, or in particular, fermented products
  • the method of the invention comprises:
  • the liquid which accompanies the cells in the suspension, or nebulization solvent is normally water and/or another chemical compound which favours the laceration process of the cell membranes to release the contents of the cell.
  • the solvent used must therefore favour the breakage of these membranes and also favour the separation of the intracellular products from the remaining solid matrix.
  • nebulization solvents are organic solutions, such as for example hydrocarbons, alcohols, esters, ethers.
  • inorganic acids can also be added to the same.
  • the nebulization of the suspension of microorganisms takes place in an expansion chamber (nebulization chamber or thermal lysing unit) containing an inert gas, water vapour, carbon dioxide and/or an organic solvent in gaseous phase, at high temperatures.
  • the nebulization chamber can already be at the lysing temperature, i.e. ranging from 120°C to 180°C, preferably from 120° C to 160° C and even more preferably from 130°C to 150°C.
  • the advantage of the present invention is, in fact, that it is possible to operate at lower temperatures with respect to those normally used in the lysing processes known in the art.
  • the interaction with a high contact surface created thanks to the nebulization, allows a rapid transfer of kinetic energy to the nebulized suspension which causes the mechanical breakage of the cell wall and/or cell membranes of the microorganisms present in the nebulized drops, by expansion.
  • the release of the intracellular contents produced by cultivation or in particular by fermentation is thus obtained, which can then be recovered along the process units following the thermal lysing unit.
  • Subsequent process units are present after the first nebulization chamber, such as, for example, accumulation tanks, cyclones or surface condensers which are kept at a progressively decreasing temperature and along which the separation of a multiphase suspension is observed, composed of the intracellular contents, solids (cell debris), an aqueous and oily liquid due to the condensation of part of the atmospheric vapours of the nebulization chamber, the latter due to the solvents which are co-fed together with the suspension of cells, and intracellular contents or lipids.
  • the first nebulization chamber such as, for example, accumulation tanks, cyclones or surface condensers which are kept at a progressively decreasing temperature and along which the separation of a multiphase suspension is observed, composed of the intracellular contents, solids (cell debris), an aqueous and oily liquid due to the condensation of part of the atmospheric vapours of the nebulization chamber, the latter due to the solvents which are co-fed together with the suspension of cells, and intracellular contents or lipid
  • the aqueous phase contains: water, residues of the cultivation or fermentation broth, salts, solvents and a few solubilized lipids.
  • the lipid or oily phase contains: organic solvents, fatty acids, lipids, water, and their salts.
  • the solid phase comprises the solid residues remaining from the cell aggregates (cell debris) which may still contain non-extracted lipids, and which can be optionally subjected to one or more additional lysing operations by subsequent nebulization operations and completion lysis in the same equipment, by recycling together with the primary feeding, to the head of the nebulization chamber, or into a subsequent lysing unit.
  • the multiphase suspension, produced in the nebulization chamber, is then subjected to separation gravimetrically or by centrifugation, in order to separate the water and solids from the oily phase containing the solvents and lipids.
  • the nature of the solvent (initial nebulization solvent) used also influences this operation as it can create phases in the form of foamy suspensions which are difficult to treat.
  • the choice of nebulization solvent must therefore also favour the separability of the components of the suspension obtained after the lysis.
  • a recovery step of the nebulization solvent is always present downstream of the separation step of the solid phases from the liquid phases, analogously to chemical processes of solvent extraction.
  • the lipids recovered can be used as such or sent to treatment destined for the final use of the same, for example within the range of use as biofuels, hydrogenation or transesterification treatment.
  • the intracellular contents of interest are separated from the solvent/aqueous phase by means of gravimetric separation or centrifugation.
  • the water can remain partly in vapour form and partly in condensed form. Water is always present, as it derives from the water present initially inside the cells.
  • the method of the invention can be used in production processes of lipids obtained as intracellular compounds in yeasts grown on sugar substrates.
  • cellular organisms such as, for example, the yeasts Lypomyces, Rhodotorula and Cryptococcus, have a cellular wall which is particularly resistant also to the actions of strong mechanical/thermal stress corresponding to pressure even higher than 1000 bar.
  • the nebulization action reduces the energy requirements of the overall process, as the heat exchange on the basis of the lysing process is favoured by the high heat exchange surface which is formed with the nebulization and cavitation phenomena induced by the high thermal profiles through the cell.
  • the environment created by the nebulization is, in fact, characterized by a high energy transfer at the cell interface, as an increase in the heat exchange between the gaseous phase and liquid phase is observed.
  • the heat exchange between gaseous phase and liquid phase is due to the fact that the interface between the two phases is greater, the smaller the diameter of the drops of liquid-solid suspension, and the transfer rate of the energy through the cell membranes increases considerably in relation to an increase in the liquid/gaseous atmosphere contact surface which is inversely proportional to the diameter of the drop and which therefore increases its efficacy by about three measurement units with respect to cases of evaporation of the liquid from a gravitational separation surface.
  • the solvents used for the nebulization and subsequent extraction of the intracellular components of yeasts, algae, bacteria and/or mildews are polar organic solvents, such as alcohols, esters, ketones, in particular methanol, ethanol, isopropanol and/or ethyl acetate are preferred, apolar organic solvents such as alkanes, in particular hexane and/or iso-octane are preferred, refinery cuts characterized by a normal boiling curve ranging from 50°C to 180°C, preferably 100°C, petroleum ether, naphthas and/or alkylated gasolines, or mixtures of the above-mentioned solvents.
  • polar organic solvents such as alcohols, esters, ketones, in particular methanol, ethanol, isopropanol and/or ethyl acetate are preferred
  • apolar organic solvents such as alkanes, in particular hexane and/or iso-octane
  • the nebulization chamber in which the suspension containing the microorganisms is effected can contain an inert gas such as, for example, nitrogen, helium, argon, carbon dioxide, in addition to water vapour, or saturated or overheated vapours of the organic solvents indicated above as nebulization solvents. This enables both the lysis of the microorganism and the extraction in the solvent of the intracellular components of interest.
  • an inert gas such as, for example, nitrogen, helium, argon, carbon dioxide, in addition to water vapour, or saturated or overheated vapours of the organic solvents indicated above as nebulization solvents.
  • the extraction of the intracellular liquids from the solid matrix of the microorganism is favoured by the pressurized solvent.
  • the extraction is then improved by a centrifugal action which favours the extraction for different densities of the solid and liquid phases of the suspension formed.
  • the temperature in the nebulization chamber is within the range of 90°C to 180°C, preferably ranging from 120°C to 160°C and even more preferably from 130°C to 150°C.
  • yeasts in particular the yeasts Lypomyces, Rhodotorula and Cryptococcus.
  • nebulization solvent with the cellular suspension before nebulization could improve the overall yield of the lysing process, as it could act chemically on the double lipid layer which forms the cell membrane, as observed in the technical solutions of the prior art.
  • a prolonged contact time between the solvent and cells to be lysed is an important factor, together with the temperature and pressure for obtaining the lysis of the microorganisms.
  • This prolonged contact time represents a further energy cost, whereas the present invention aims at reducing these costs and is therefore characterized by low contact residence times of the solvent with the cells to be lysed, both when the solvent is put in contact with the cells before being introduced into the nebulizer, and also when the solvent is present in the nebulization chamber where the suspension of microorganisms to which the solvent has not been previously added, has been nebulized.
  • the dimensioning of the nebulization volume for the method at the basis of the present invention arises from the application of the concept of separation of the suspension containing the solid-liquid phases, deriving from the cell lysing action, from the vapours released after nebulization.
  • This concept also envisages the use, necessary at times, of various separation chambers, arranged in series with respect to the gas flow, which are necessary for favouring the separation of the cell contents in addition to the solid residues of the lysed cell.
  • the form of the nebulization chamber must favour the separation from the vapours of the suspension and the outflow and collection of the latter from the bottom of the same.
  • Cylindrical forms also sandwiched by gridded or lamellar velocity reducers, with a truncated-conical terminal for collecting the suspensions, are preferred forms of the nebulization chamber.
  • the thermal profile along the process line which starts from the nebulization volume, which is defined by the heat exchange effected at the wall or by means of exchange inserts in the separator volumes, generally influences, on the other hand, the release of solvents from the suspension deriving from the lysis.
  • This profile is defined by the flow-rate of the gases released and entrainment permitted of the cell contents due to the physico-chemical liquid/vapour equilibrium and action of the gas velocities.
  • the nebulization chamber can already be at the lysing temperature or at higher temperatures in order to favour high treatment flow-rates in the lysis, and contains the above-mentioned gases or vapours at a temperature equal to or higher than the temperature at which the lysis and extraction are to be effected.
  • the suspension containing the microorganisms to be lysed can enter the nebulization chamber at a lower temperature than that at which the lysis is to be carried out.
  • the solvent enters with a flow and thermal level which are such as to reach, together with the substance to be lysed, the lysis conditions in the nebulizer.
  • a possible increase in temperature in the separation volumes, subsequent to the nebulization chamber, has a negative effect on the lipids, increasing the quota of fatty acids contained in the suspension. This increase, however, can favour the extraction from the solid matrix.
  • the intracellular components of interest of a lipid nature such as triglycerides, mono and diacyl glyceride fatty acids, phospholipids, phytols, cholesterols, are released in a suspension of high-boiling oils (lipids have a boiling point at room temperature higher than 300°C) together with the solid cell residues.
  • the suspension of high-boiling oils is then separated from the nebulization solvent, if this can be separated from said oils, such as, for example when the solvent is aqueous or polar, and is sent to separation of the solid cell residues (debris); as already previously pointed out, this separation can be effected gravimetrically, by density, centrifugation or filtration, for example with a hydrocyclone, decanter or tricanter or by distillation, by means of membranes or by evaporation of the product from the nebulization/extraction solvent.
  • the water present in the expansion chamber of an intracellular origin or deriving from the suspension of cells or from the high-temperature vapour possibly added for purposes of heat exchange, together or separately with the suspension to be lysed, is preferably removed from the suspension to facilitate the subsequent solid/liquid separation, avoiding foams and suspensions which negatively interact on the subsequent separation of the cell products.
  • the nebulization solvent is optionally fed together with pressurized water vapour in order to control the lysing temperature in the nebulizer. This operative methodolgy is used if there is an easy water/solvent separation in liquid phase. In these cases, the vapour, containing the solvent and water, is sent to a second chamber and once separated from the water, is recycled to the nebulizer, where it is integrated with fresh solvent.
  • the equipment which can be used for the nebulization is basically analogous to or the same as that used for the nebulization operation of a liquid suspension of a solid (spray-dryer).
  • a spray-dryer is a combination of apparatuses composed of a feeding pump of a liquid containing a suspension, an inorganic solid or not, a gas or vapour source such as a thermal and mechanical energy carrier, an atomizer, a heater, a dispersion space of the vapour/liquid/solid mixture leaving the atomizer, or drying chamber, in addition to treatment/condensation systems of the vapour/exhaust gas mixture and recovery of possible powders.
  • the solid compound is extracted dry at the temperature of the drying chamber.
  • the materials to be used for lysing applications must be defined in relation to the operating conditions, i.e. in relation to the nominal functioning temperature and pressure, and in relation to the resistance to the aggressiveness conditions of the environment in turn in relation to the solvents and contents in the liquids which accompany the cells.
  • the application method, object of the present patent application allows considerable savings on the variable and CAPEX costs (Capital expenditure) of the plants for effecting lysing operations of cells and extraction of the intracellular compounds, due to the fact that, thanks to the method of the invention, there is a significant energy saving, as apparatuses for thermally or mechanically lysing the cells in a first phase with subsequent costly extraction units of the lipid, are no longer necessary.
  • the objective is only to define the lysing capacity of the process proposed.
  • the effective lysis is then evaluated a posteriori with classical analyses by means of a Soxhlet liquid-solid extraction unit.
  • a MINI Spray Dryer 190 of the supplier BUCHI was used, which, as a dryer, was capable of evaporating about 100-500 g/h of water with a flow of N 2 of 700-1,000 Nl/h which can be heated to a maximum temperature of 250°C, and which is therefore capable of producing about 1-100 g/h of dried powder having a particle-size in the order of 0.2-10 microns.
  • the overall time used by the biomass for following the "hot part" of the circuit as far as the nebulizing nozzle was 10-30 seconds.
  • the whole circuit of gas fed to the nebulization chamber as far as the nebulization or spray chamber, is under a slight vacuum, indicatively 0.97 atm.
  • Nitrogen was present as inert gas in the nebulization chamber in which the suspension to be treated was sprayed, whose temperature was maintained at approximately the same value as that at the inlet of the cyclone separator (temperature to which the sample is immediately brought after the nozzle by evaporation of the suspension water and which was constantly monitored as indicated in Table 1).
  • a Soxhlet unit was used for the extraction of the lipids from the cellular solids.
  • a paper thimble was washed with the extraction solvent and then brought to a constant weight in an oven at 105°C.
  • the solid to be treated (3-20 g) was charged into the thimble.
  • the extraction solvent (150-200 ml) was charged into the flask, whose tare-weight had been accurately determined.
  • the Soxhlet was substituted with a claisen, and the solvent was distilled, first at atmospheric pressure and then under vacuum at about 0,005 absolute atm. The lipid extract remains in the flask.
  • thermobalance Perkin Elmer
  • FT-IR Fourier transform spectrophotometrical analyses
  • the test SD100 was carried out with a diluted feeding of about 1:3 in demineralized H 2 O with respect to the others.
  • the three tests differ in the temperature of the nebulization chamber (T out in Table 1 below) and in particular 95°, 130° and 150° C.
  • T out in Table 1 the temperature of the nebulization chamber
  • the Cryptococcus used for the test SD104 indicated in the table before drying in the spray dryer, was subjected to diafiltration with the addition of demineralized water and ultrafiltration to separate/collect the yeast cells and leave the sugars present initially with the yeast in water, in order to remove the residual glucose of the fermentation.
  • Table 1 - nebulization conditions were subjected to diafiltration with the addition of demineralized water and ultrafiltration to separate/collect the yeast cells and leave the sugars present initially with the yeast in water, in order to remove the residual glucose of the fermentation.
  • TEST SUSPENSION weight (g) dry weight (dw%) SD100 Lypomyces 150 9.8 SD101 Lypomyces 19 29.5 SD102 Lypomyces 67 29.5 SD103 Rhodotorula 26.8 13.0 SD104 Cryptococcus 127.2 4.2 (continuation of Table 1)
  • TEST SPRAY CONDITIONS estimate recovery (dw%) nitrogen (l/h) Vacuum atm Set point Pump T in (°C) T out (°C) f.
  • T in (°C) temperature of the nitrogen at the inlet of the nebulization chamber, therefore before introduction of the suspension to be nebulized
  • T out (°C) temperature of the nebulization chamber after the introduction
  • the yeasts collected in the cyclone positioned after the nebulization chamber prove to be dried, as they have a residual water content of less than 5%. These dried yeasts were characterized by means of thermogravimetric analyses. Thermogravimetry consists of a registration of the evolution of the weight in air between room temperature and 930°C, with a gradient of 10°C/min. The samples subjected to thermogravimetry are those listed in Table 2 below.
  • the normal nebulization operation of the present instrument used as spray dryer up to a temperature of 95°C produces an effective drying of the suspension of these yeasts (the residual water in the solids is less than 5%) but is not effective in contemporaneously effecting a the lysis of the cell membrane.
  • Figure 1 indicates the extraction yield (dw%) of the lipids, in relation to the nebulization temperature of the suspensions of Lypomyces (L), Rhodotorula (R) and Cryptococcus (C) yeasts.
  • FIG. 1 shows the thermograms which indicate the results obtained with the method of the invention.
  • figure 2 shows a comparison between thermograms of Lypomyces yeast cells after nebulization at increasing nebulization temperatures, i.e. at 95°C, 130°C and 150°C, but before solvent extraction of the lipids contained therein.
  • thermograms of figure 2 represent the residual weight % curves (left scale) and derivative curves (D%/DT) obtained by heating the nebulized samples in an atmosphere of air with a heating profile of 10° C/min at temperatures of 95°C, 130°C and 150°C.
  • the temperature at which the main weight loss is observed in the thermogram is within the range of 280-380°C.
  • This weight loss indicates the breakage of the membrane and is correlated, in the graph, with the temperature at which the sample has been nebulized.
  • T out 95°C
  • the sample SD101 at 130°C and the sample SD102 at 150°C leads to a lower temperature with respect to the weight loss (breakage of the membrane) of the sample analyzed.
  • Figure 3 shows the thermogram of a suspension of Lypomyces yeast dried in an oven at 105°C, from which it can be deduced that the weight loss, i.e. the measurement of a hypothetical lysis due to the thermal drying effect alone, does not take place at these temperatures which are those normally used in the drying technology of a spray-dryer. Only with much higher temperatures, and longer treatment times by a few orders of magnitude with respect to approximately ten microseconds, can the thermal lysis lead to breakage of the membranes of the yeast used in the example of nebulising.
  • thermograms of Figure 2 also show how the residual humidity content in the samples produced with nebulization was lower than 5% (weight loss at a temperature of 105°C, less than 5%) and how there was a modest ash content, less than 4% (residual weight at a temperature of 600°C). The beginning of the significant degradation phenomena is indicated by the weight loss which is observed at about 220°C.
  • the weight loss between 280°C and 380°C begins at lower temperatures with an increase in the nebulization temperature of the sample, which indicates a greater facility in releasing the endocellular substances towards the outside environment.
  • the other weight losses observed, in particular at about 250°C, 420°C and 500°C, which are other cell components, such as for example, carbohydrates, did not seem to be influenced, on the contrary, by the nebulization treatment to which the sample was subjected.
  • Figure 4 shows the curve of the IR spectrum of the extract SOX107 (at 150°C) ( fig. 4b ) (again with reference to the samples indicated in Tables 1 and 2), and for direct comparison, the IR spectrum of a reference triglyceride, i.e. with triolein ( fig. 4a ). From a comparison, it can be deduced that the spectra obtained with the method of the invention are those of triglycerides, with the band characteristic of carbonyl at 1750 cm -1 . There is also the presence of free fatty acids (band at 1705 cm -1 ) deriving from the hydrolysis of the lipids (triglycerides).
  • a plant scheme is illustrated below for the use of the method of the invention, wherein the lipids contained in a yeast of the Rhodotorula family, were extracted.
  • the substance to be lysed, in suspension in the fermentation water is treated in lysis together with n-hexane as solvent, necessary for the extraction of the lipids from the solid.
  • Figure 5 represents the plant scheme of a closed-cycle solution wherein the nebulization is obtained with a usual industrial spray-dryer, in which the specifications of the operations are in line with the data listed in Example 1 and vary according to the product to be lysed, in relation to the temperature conditions and solvent used.
  • a wide range of nebulization chambers and injection systems in line with the specific spray-drying technology are available in the catalogue.
  • Table 3 below provides a key of the symbols of the plant scheme of figure 5 :
  • Table 3 E-100 E-101 Tube-bundle heat exchangers q100 Energy supplied for the heating by means of an external service fluid VLV-100 Lamination valve MIX-101 Sprayer or nebulizer V-100 Nebulization chamber E-102 Heat exchanger with wall vapour of V-100 C-500-2 Column for water-solvent separation X-100 Oil-solid lipid separator MIX-102 Filling process of the solvent P-100 Solvent entrainment pump RCY-2 Recycling
  • the scheme represented in figure 5 is composed of a pump P-100 which carries a solvent for the extraction of the intracellular product, under pressure at an adequate value for the heat exchange and lysing operation in the nebulization chamber.
  • a specific temperature for the cell disruption of the microorganism and a residence time of the nebulized suspension within the range of 5-300 sec, preferably about 10 seconds, must be obtained.
  • the liquid passes through an exchanger without changing phase, and then enters a static grid mixer where it encounters and entrains the suspension or turbid solution to be lysed (flow "1") to a spray-dryer (nebulizer).
  • the turbulent radial mixing mechanism in the mixer reduces the radial velocities and dimensions of the monophase aggregates to be mixed, increasing the contact surface and stimulating the thermal and chemical concentration exchange, improving the mixing.
  • the length of the dispersers necessary depends on the contact time required. For mass transfer processes in which the equilibrium is rapidly established, a length of 5 diameters is generally sufficient.
  • the nebulization of the products is effected with various spray-dryers, under controlled temperature and air-flow conditions, which can be rotating for an improved homogenization of the chamber V-100.
  • the suspension is divided into nebulized drops which then come into contact with water vapour in the present example for the temperature control of the nebulization chamber V-100 and expands.
  • the temperature of this chamber is kept above the lysis threshold by a flow of additional and wall vapour.
  • the vapours evacuate the chamber V-100 under controlled temperature and flow conditions.
  • the suspension of solid residues and lipids is continuously discharged, by gravity and pressure, from the nebulization chamber, and can be sent to separation of the cell residues (this is the flow indicated with "4" in the scheme in figure 5 ).
  • the flow "2" represents a flow of water vapour for the temperature control of the nebulization chamber.
  • vapours After cooling to below 100°C, the vapours are sent to a recovery column of the solvent, C-100, which is then recycled together with a make-up flow to the pump P-100.
  • the water separated (streams "6" and "7") is sent to a treatment plant before being disposed of.
  • Table 4 provided in figure 6 indicates the material and energy balance of the example relating to a Rhodotorula treated via sugar fermentation.
  • the scheme was developed for a concentration of solids to be lysed at 30% by weight in the turbid solution to be treated.
  • the specification conditions for the lysing operation are indicated in Table 4. In this respect, 1.52 kg of vapour are necessary at 30 bar and 21 kg of cooling water per kg of turbid solution to be lysed. The other exchanges are compensated internally.
  • the extraction operation is then effected on a tricanter using hexane.

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Claims (16)

  1. Procédé de récupération de composants intracellulaires à partir d'une suspension de microorganismes, lequel procédé comporte les étapes suivantes :
    - alimenter un nébuliseur avec au moins deux courants envoyés simultanément, étant entendu que le premier courant comprend la suspension aqueuse de microorganismes cellulaires à lyser, et que le deuxième courant comprend un solvant liquide ou gazeux ;
    - nébuliser ladite suspension dans une chambre de nébulisation maintenue à une température valant de 120 à 180 °C, de manière à provoquer la lyse des parois cellulaires des microorganismes et à ce qu'il se forme une suspension contenant les composants intracellulaires et les résidus de cellules issus de la lyse, ainsi qu'une phase vapeur comprenant le solvant organique ;
    - et séparer les composants intracellulaires d'avec les résidus de cellules issus de la lyse.
  2. Procédé conforme à la revendication 1, dans lequel le solvant est un solvant organique polaire.
  3. Procédé conforme à la revendication 2, dans lequel le solvant organique polaire est choisi parmi les alcools, les esters, les cétones et les mélanges de tels solvants.
  4. Procédé conforme à la revendication 3, dans lequel le solvant organique polaire est choisi parmi le méthanol, l'éthanol, l'isopropanol et/ou l'acétate d'éthyle.
  5. Procédé conforme à la revendication 1, dans lequel le solvant est un solvant organique apolaire.
  6. Procédé conforme à la revendication 5, dans lequel le solvant organique apolaire est un alcane.
  7. Procédé conforme à la revendication 5, dans lequel le solvant organique apolaire est choisi parmi l'hexane et/ou l'iso-octane, les coupes de raffinerie dont l'intervalle d'ébullition dans les conditions normales va de 50 °C à 180 °C, de préférence 100 °C, l'éther de pétrole, les naphtas et/ou les essences alkylées, et leurs mélanges.
  8. Procédé conforme à la revendication 1, dans lequel la nébulisation de la suspension de microorganismes cultivés se déroule dans une chambre de nébulisation contenant un gaz inerte, choisi parmi de la vapeur d'eau, du dioxyde de carbone ou le solvant de nébulisation à l'état gazeux.
  9. Procédé conforme à la revendication 1, dans lequel la température régnant dans la chambre de nébulisation vaut de 120 à 160 °C, et de préférence de 130 à 150 °C.
  10. Procédé conforme à la revendication 1, dans lequel les microorganismes sont des levures, des algues, des bactéries et/ou des moisissures.
  11. Procédé conforme à la revendication 10, dans lequel les microorganismes cultivés sont des levures choisies parmi les genres Lipomyces, Rhodotorula et Cryptococcus.
  12. Procédé conforme à la revendication 1, dans lequel les composants intracellulaires sont des lipides intracellulaires produits par les microorganismes cultivés, par suite d'une fermentation de sucres.
  13. Procédé conforme à la revendication 1, dans lequel la séparation des composants intracellulaires d'avec les résidus de cellules issus de la lyse est opérée par gravimétrie ou par centrifugation.
  14. Procédé conforme à la revendication 1, dans lequel la suspension contenant les composants intracellulaires et les résidus de cellules issus de la lyse est soumise à au moins une opération de lyse supplémentaire, par recyclage de cette suspension.
  15. Procédé conforme à la revendication 14, dans lequel on recycle la suspension contenant les composants intracellulaires et les résidus de cellules issus de la lyse en la faisant rejoindre la suspension initiale à lyser, en amont de la chambre de nébulisation.
  16. Procédé conforme à la revendication 14, dans lequel on recycle la suspension contenant les composants intracellulaires et les résidus de cellules issus de la lyse en l'envoyant vers une unité de lyse, en aval de la chambre de nébulisation.
EP13826947.7A 2012-12-20 2013-12-11 Procédé de récupération de composants intracellulaires à partir de micro-organismes fermentés Active EP2935561B1 (fr)

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IT002195A ITMI20122195A1 (it) 2012-12-20 2012-12-20 Metodo per il recupero di componenti intracellulari da microorganismi fermentati
PCT/IB2013/060813 WO2014097057A1 (fr) 2012-12-20 2013-12-11 Procédé de récupération de composants intracellulaires à partir de micro-organismes fermentés

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CA2303399A1 (fr) 1997-09-23 1999-04-01 Ib2, L.L.C. Procede et dispositif de traitement par cycle thermique rapide
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DE102006042161A1 (de) 2006-09-06 2008-03-27 Ecoenergy Gesellschaft Für Energie- Und Umwelttechnik Mbh Verfahren und Vorrichtung zur Trennung von fossiler und nativer Organik aus organischen Stoffgemischen
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ITMI20122195A1 (it) 2014-06-21
BR112015011696B1 (pt) 2022-05-31
CN105073975B (zh) 2018-08-21
EP2935561A1 (fr) 2015-10-28
BR112015011696A2 (pt) 2017-07-11
ES2627948T3 (es) 2017-08-01
CN105073975A (zh) 2015-11-18
WO2014097057A1 (fr) 2014-06-26
US20150299096A1 (en) 2015-10-22
US9593067B2 (en) 2017-03-14
PL2935561T3 (pl) 2017-08-31

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